Multiple Single-Nucleotide Polymorphism Detection for Antimalarial Pyrimethamine Resistance via Allele-Specific PCR...

Area of Science:

• Molecular diagnostics
• Biotechnology
• Parasitology

Background:

• Antimalarial drug resistance (ADR) is a major threat to malaria control.
• Molecular genotyping for ADR detection is complex and requires expensive equipment.
• There is a critical need for rapid, point-of-care testing (POCT) molecular tools for field use.

Purpose of the Study:

• To develop and validate a novel POCT platform for detecting single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (pfdhfr) gene.
• To assess the utility of this platform for identifying pyrimethamine resistance-related mutations in clinical samples.

Main Methods:

• A POCT platform combining multiple allele-specific PCR (AS-PCR) with a gold nanoparticle (AuNP)-based lateral flow biosensor was designed.
• The AS-PCR employed modified primers for enhanced specificity.
• Detection involved biotin and digoxin-labeled amplicons captured by streptavidin-AuNPs and anti-digoxin antibodies, forming a visible gold-red line.

Main Results:

• The platform successfully detected SNPs (N51I, C59R, S108N) in the pfdhfr gene of 98 clinical Plasmodium falciparum isolates.
• Compared to Sanger sequencing, overall sensitivity was 97.96% and specificity ranged from 95.92% to 100%.
• The limit of detection was as low as 100 parasites/μl in blood filter paper samples.

Conclusions:

• The developed POCT platform provides a sensitive, specific, and rapid method for molecular surveillance of antimalarial drug resistance.
• This technology is easily adaptable for detecting SNPs related to other infectious and genetic diseases.
• It offers a valuable tool for decentralized malaria diagnosis and resistance monitoring.